Many extracellular stimuli produce their physiological effects through promotion of inositol lipid hydrolysis. These responses historically have been thought to occur either through heptahelical receptors coupled to heterotrimeric G proteins that activate phospholipase C-beta (PLC-beta) isozymes or through tyrosine kinase-activating receptors producing SH2 domain-dependent activation of PLC-gamma. However, small GTP binding proteins of the Rac family activate PLC-beta2, and we recently illustrated that this interaction occurs through GTP-dependent binding of Rac to the PH domain. PLC-epsilon was discovered as a novel phospholipase C activated by heterotrimeric G protein subunits (Galpha12/13 and Gbetagamma) as well as by interaction of Ras family GTPases with two Ras-associating domains in the C-terminus. We recently discovered that Rho family GTPases also directly activate PLC-epsilon in part through a unique insert in the conserved catalytic core of the enzyme. In addition PLC-epsilon acts as an upstream activator of small GTP binding proteins through guanine nucleotide exchange activity of a Cdc25 domain in its N-terminus. Thus, signals from both heterotrimeric and small GTPases converge on PLC-beta2, and both converge and diverge through the nexus PLC-epsilon. The goal of this research is to elucidate the mechanistic/structural bases for the regulated activities of PLC-beta and PLC-epsilon isozymes. We are defining the spectrum of small GTPases within the Rho and Ras families that bind to and activate PLC-epsilon and PLC-beta2. Consequently, we will delineate the functional interfaces responsible for these interactions by mutational analyses (Specific Aim 1). These studies will include surface plasmon resonance analyses to define direct interactions, co-transfection studies measuring inositol phosphate accumulation, and in vitro reconstitution approaches quantifying phospholipase C activity. PLC-epsilon exhibits novel downstream signaling activities through the functional interplay of its Cdc25 domain with other portions of PLC-epsilon. The multifunctional nature of this isozyme will be explored in detail (Specific Aim 2) by quantitatively assessing the guanine nucleotide exchange potential of PLC-epsilon against a broad panel of GTPases and by mutationally defining the interacting surfaces of PLC-epsilon and Ras GTPases. An ultimate goal of our research is to define the structural organization of PLC-beta and PLC-epsilon using crystallography. Accordingly, Specific Aim 3 will focus on structure determination of (i) full length and various structural domains of PLC-beta and PLC-epsilon isozymes, including high affinity complexes between: (ii) Rac and the PH domain of PLC-(2, (iii) Ras family GTPases and portions of PLC-epsilon containing the Ras-binding or Cdc25 domains, as well as the newly identified Rho-interacting region within the catalytic core. Accomplishment of these research goals will provide major new insights into the underlying mechanistic and structural bases for the diverse functions of these multifaceted signaling proteins that fulfill central roles in the signaling responses to many extracellular stimuli and that exist as potential drug targets for broadly diverse diseases.